Kaposi's sareoma(KS)is the most common malignancy associated with HIV infection. Human herpesvirus-8(HHV-8)1 Kaposi's sarcoma-associated herpesvirus , is consistently detected in the complex cellular lesions comprising KS. Viral genes are expressed in endothelial cells, in spindle cells of probable endothelial origin and in monocytes. The infected endothelial cells synthesize genes that include HHV-8 encoded homologs of human growth regulatory proteins, whereas monocytes appears to be productively infected. HHV-8 has also been found in the malignant B cells that make up body cavity based lymphomas(BCBLs). In BCBLs, the circular HHV-8 genome persists in latently infected B cells, that in contrast to KS derived cells grow out readily in vitro. Although HHV-8 has been detected in endothelial cells, B cells and monocytes derived from patient samples, transmission of virus isolated from multiple sources to cells in vitro has proved to be extremely inefficient - permitting detection by PCR alone. The events accompanying viral entry have not been described and persistence of virus in infected cells consistent with progression to latency, has not been well documented. Our preliminary work indicates that FITC-HHV-8 binds human dermal microvascular endothelial cells (MVDEC). Attachment of HHV-8 is followed by internalization and transport to the nucleus. During the first 24 hrs viral episomes can be detected by Gardella analysis, followed by rapid linearization of most viral DNA, consistent with a productive/abortive infection. HHV-8 transcripts can be detected by PCR and by RNA blot hybridization, suggesting infection of MVDEC is more efficient than previously described. Studies in progress show some viral transcripts are present at l0 days, raising the possibility that after initial lytic infection, latent virus may sometimes persist. We now propose pilot studies to examine the biology of HHV-8 in MVDEC. First, we will optimize the conditions of virus and cell growth required for efficient infection. Second, using standard molecular/immunologic techniques, we will delineate which viral genes transcripts are expressed early after infection and will determine whether virus infection persists in a subpopulation of cells. If so, the pattern of HHV-8 expression in persistently infected cells will be determined. Third, we will analyze how virus enters MVDEC and identify the structures that appear to be involved by confocal microscopy and transmission EM. Fourth we will determine which protein(s) are involved in attachment of HHV-8 to the cell surface through synthesis of mabs to puritied virions that neutralize infection. Conversely, we will identify cell surface proteins required for attachment of HHV-8 by generating mabs to MVDEC and identifying antibodies that prevent virus binding. The information and reagents developed by these pilot experiments will provide a basis for future studies to delineate how this newly discovered herpes virus gains access to human cells and expresses viral gene products that may lead to cellular immortalization.